Study on Purification of Cellulase from Penicillium sp. and Pretreatment of Cellulosic Biomass

纤维素生物质是地球上最丰富的可再生资源，其资源化或能源化利用是目前国内外研究的热点。蔗渣作为一种大宗糖业固体废弃物，其开发利用正受到越来越多的关注。 Penicilliumsp.T24-2是一株降解蔗渣能力较强的丝状真菌，其固态发酵所产粗酶液，经阴离子交换柱DEAE-SepharoseFastFlow层析后，分离出两种β-葡萄糖苷酶。通过SDS-PAGE凝胶电泳分析，确定其分子质量约分别为20kDa、35kDa。已纯化的两种β-葡萄糖苷酶均为单亚基蛋白，其酶促最适反应温度分别为60℃、45℃；最适酶促反应pH值分别为pH4.5、pH5.0。 对3,5-二硝基水杨酸（DNS）试剂测定还原糖方...Cellulosic biomass is the most abundant renewable resource on the earth. Bagasse is a kind of bulk solid waste of sugar industry which has been received more and more attention recently. The strain Penicillium sp.T24-2 was isolated and preserved in this lab which could produce cellulases at a high level by solid-state fermentation. Two kinds of β-glucosidase were isolated by anion exchange DE...学位：理学硕士院系专业：生命科学学院生物学系_微生物学学号：2172006115214

木聚糖酶原位协同水解预处理大米草的研究

实验主要研究了氨水/双氧水结合球磨预处理对重组木聚糖酶协同水解大米草的增强效果。首先使用氨水与双氧水预处理大米草，预处理物与原料大米草随后用于球磨。然后将预处理底物分别用于结构表征及木聚糖酶的协同酶解。结果说明氨水结合双氧水预处理能有效脱除大米草中的木质素(63.81%)，球磨处理能对纤维素结晶结构有所破坏，两者有效提高木聚糖酶的水解率。而经预处理过的大米草的酶水解物主要成分为木糖(12.54%)，木二糖(40.38%)，及少量阿拉伯糖(5.50%)。以上结果说明碱预处理结合球磨预处理对后续木聚糖酶协同水解大米草产木寡糖具有明显的促进作用。厦门大学能源发展基金(2018NYFZ03

Thermal Degradation of Bamboo Lignin in Alkaline Ethanol Solvent and Product Analysis

本研究以nAOH-乙醇水溶液为溶剂体系对竹木质素进行热降解,主要考察了nAOH浓度、反应温度、反应时间、乙醇用量等条件对竹木质素降解转化为酚类化合物的影响。通过gC-MS及fT-Ir对降解产物进行分析检测,得出最佳反应条件为:竹木质素5 g,nAOH浓度(基于乙醇水溶液)20 g/l,乙醇10 Ml,反应时间2 H,反应温度240℃。在此条件下,降解产物中总的酚类化合物的相对峰面积为73.88%,残渣率为30.67%。竹木质素的降解主要产物是酚类化合物:苯酚(17.98%)、2-甲氧基苯酚(16.49%)及1,2-苯二酚(10.03%)。与现有文献相比,本文竹木质素在碱性乙醇溶剂体系中降解能够获得较高产量的酚类化合物,有望实现竹木质素的高值化利用。The thermal degradation of bamboo lignin in NaOH-ethanol solvent system was studied in this work.The effects of NaOH concentration, reaction temperature, reaction time, and dosage of ethanol on the phenolic products were investigated, respectively.The degradation products were analyzed by GC-MS and FT-IR.The optimum conditions were achieved as follows: mbamoo lignin = 5 g, CNaOH(in alcohol aqueous systems) = 20 g/L, Vethanol = 10 mL, reaction temperature is 240 oC and reaction time is 2 h.The total relative peak area of phenolic products in the degradation products was 73.88%, and the yield of residue was 30.67%.The main products of bamboo lignin degradation were phenols, such as phenol(17.98%), 2-methoxyphenol(16.49%) and 1,2-benzenediol(10.03%).The results indicated that higher yield of phenolic products was obtained under the optimized conditions compared with the reports.This work showed the potential to degrade bamboo lignin into high value products in NaOH-ethanol solvent system.农业部“引进国际先进农业科学技术”项目(2013-Z70); 福建省自然科学基金(2012J05029

Optimization and characterization of sodium carboxymethyl cellulose with a high degree of substitution prepared from bamboo shavings

从竹材加工剩余物竹屑中提取纤维素,然后制备羧甲基纤维素钠。采用PlACkETT-burMAn(Pb)设计与中心组合法对影响竹纤维羧甲基纤维素钠取代度的6个相关因素进行了研究,结果表明,关键因子为氢氧化钠与纤维素的质量比、氯乙酸钠与纤维素的质量比和醚化时间;建立此三因素对羧甲基纤维素钠取代度影响的二次回归模型,优化得到最佳制备条件是:氢氧化钠与纤维素的质量比为0.8、氯乙酸钠与纤维素的质量比为1.2,醚化时间为2 H。制得羧甲基纤维素钠的黏度为136 M PA·S,取代度为0.93。利用扫描电镜、红外光谱、X射线衍射和热重分析等对制得样品进行表征。结果表明,利用廉价的竹屑可以制备高取代度的羧甲基纤维素钠。The preparation of carboxymethyl cellulose( CMC) with a high degree of substitution( DS) using cellulose from bamboo residues is investigated.The factors affecting DS of CMC are optimized by Plackett-Burman Design( PB) and Central Composite Design.The results of PB indicate that the mass ratio of Na OH and cellulose,the mass ratio of Cl CH2 COONa and cellulose,and the etherification time are the key factors.The quadratic regression model of these three factors is established.The optimal conditions are shown as follows: 0.8 of the mass ratio of Na OH and cellulose,1.2of the mass ratio of Cl CH2 COONa and cellulose,2 hours of the etherification time.Under this condition,CMC with a DS of 0.94 and viscosity of 136 m Pa·s is obtained.The CMC is characterized by SEM,FT-IR,XRD,and TGA.It indicates that cheap bamboo shavings could be used to prepare CMC with a high DS.国家自然科学基金(21303142;31170067); 福建省中青年教师教育科研项目(JA14010); 厦门市海洋经济发展专项资金项目(14GZP59HJ29

Synthesis and characterization of cellulose acetate with high degree of substitution from miscanthus

以芒草为原料,用Na OH/H_2O_2溶液体系预处理制备芒草纤维,在冰醋酸环境下,以浓硫酸为催化剂与醋酸酐酯化制备芒草醋酸纤维素。优化了预处理条件:温度、时间、次数和酯化条件:催化剂量、温度、时间、醋酸酐量,最佳条件下制备出的芒草纤维的纤维素、半纤维素和木质素的质量分数分别为75.3%、17.3%、5.1%,制备出芒草醋酸纤维素的取代度DS=2.8,特性黏度[η]=1.24 d L/g,达到美国联邦贸易委员会指南认定的三醋酸纤维素标准。利用扫描电镜(SEM)和热分析(TG、DSC)对制得样品进行表征。结果表明,可以利用Na OH/H_2O_2水溶液体系预处理芒草原料制备芒草纤维,并进一步酯化制备出高取代度的醋酸纤维素。A procedure for synthesizing cellulose acetate with high degree of substitution from miscanthus biomass is developed. The miscanthus fiber is prepared by pretreatment of miscanthus biomass with Na OH / H_2O_2,which is then reacted with acetic anhydride in an acetic acid solvent to synthesize cellulose acetate by using concentrated sulfuric acid as catalyst. The effects of the pretreatment factors( such as pretreatment temperature,time and number of times) and the esterification factors( such as catalyst volume,reaction temperature,reaction time and acetic anhydride volume) are studied. Under the optimal conditions,the contents of cellulose,hemicellulose and lignin for prepared miscanthus fiber are75. 3%,17. 3% and 5. 1%,respectively. The degree of substitution( DS) and the intrinsic viscosity( [η ]) of the obtained miscanthus cellulose acetate are 2. 8 and 1. 24 d L / g,respectively. The miscanthus biomass,fiber and cellulose acetate are characterized by SEM,TG and DSC. This study shows that cellulose acetate with high degree of substitution can be prepared from miscanthus biomass pretreated with Na OH / H_2O_2.国家自然科学基金(21303142;31170067);; 福建省中青年教师教育科研项目(JA14010);; 厦门市海洋经济发展专项资金项目(14GZP59HJ29);; 福建省海洋高新产业发展专项项目(闽海洋高新[2014]25号);; 厦门大学校长基金(20720150090

羧甲基纤维素-丁苯橡胶复合黏结剂在硅基锂离子电池中的应用

黏结剂是影响锂离子电池性能的重要因素之一,将羧甲基纤维素(CMC)和丁苯橡胶(SBR)联合使用,有利于缓解锂离子电池电极的体积效应,但SBR的导电性较差不利于提升电池的电化学性能.为此,制备了不同取代度(0.23~0.86)的低聚合度CMC,并和4种高聚合度CMC分别应用于硅负极,进行循环性能的比较;采用低聚合度CMC-SBR制备电极,进行电化学性能测试.结果表明,采用低聚合度CMC制备的电极的电化学性能优于高聚合度CMC,且不添加SBR时表现出相对优异的电化学性能,其中取代度为0.55的低聚合度CMC制备的电极循环性能最佳.因此,使用低聚合度CMC时不需添加SBR,既减少了用料成本,又节约了电池内部空间,有利于增加有效的电极材料.厦门大学校长基金(20720150090);厦门大学能源发展基金(2017NYFZ02

Research Progress of α-Glucuronidase, an Enzyme for Degrading Hemicellulose Side-Chain

半纤维素是自然界中最丰富的可再生资源之一,将半纤维素降解为单糖并转化为燃料或化学品一直是科学界研究的热点。半纤维素是由木糖基主链以及α-葡萄糖醛酸等侧链共同组成的异质多聚体。α-葡萄糖醛酸酶是半纤维素完全降解过程中的关键酶之一,能够水解4-O-甲基葡萄糖醛酸与木糖之间的α-1,2-糖苷键。本文综述了α-葡萄糖醛酸酶的分类、催化机制及晶体结构、酶学性质和基因克隆表达等方面的研究进展,同时对该研究进行了展望。Hemicellulose is one of the most abundant renewable resources in nature.The bioconversion of hemicellulose into biofuels or chemicals is a research hotspot in the world.Hemicellulose consists of a backbone of xylan residues and some branches like glucuronic acid.α-Glucuronidase, which is capable to hydrolysis the α-1,2-glycosidic bond between xylan and glucuronic acid, is one of the key enzyme to degrade hemicellulose completely.The recent research progresses on catalysis mechanism, structure, charaterization, and gene cloning of α-glucuronidase are summarized in this paper.国家自然科学基金(31170067;21303142); 国家重点基础研究发展计划(973计划;2010CB732201); 福建省自然科学基金(2012J05029); 农业部“引进国际先进农业科学技术”项目(2013-Z70

Cloning and heterologous expression of a novel GH10 xylanase gene from Hypocrea orientalis EU7-22

木聚糖酶是降解半纤维素最主要的酶,对于开发可再生生物能源具有重要的应用价值。分别以东方肉座菌(HyPOCrEA OrIEnTAlIS)Eu7-22的基因组dnA和C dnA为模板,利用染色体步移和PCr技术首次克隆获得该菌gH10家族木聚糖酶Ⅲ的基因(XynⅢ),并对其进行生物信息学分析。结果表明:该基因全长1283 bP(gXynⅢ),含有3个内含子;CdS序列为1044 bP(CXynⅢ),编码347个氨基酸,n端含有一个16 AA的信号肽序列;XynⅢ氨基酸序列与TrICHOdErMA PSEudOkOnIngII的EndOXylAnASE具有较高的同源性。经生物信息学分析,XynⅢ成熟蛋白可能含有18个n-糖基化位点,其理论等电点(P I)为6.14,蛋白质分子质量为36.55 ku,属于亲水性蛋白;SWISS-MOdEl建模预测,XynⅢ成熟蛋白中含有11个α螺旋,其核心结构为8个β折叠片围成一个柱状结构。同时将编码成熟蛋白的基因片段MXynⅢ与P PIC9k质粒连接构建表达载体后转化毕赤酵母,对重组子表达产物进行酶活检测显示该基因能在毕赤酵母中表达有生物活性的XynⅢ并分泌到胞外,发酵液中的木聚糖酶活在诱导培养168 H后可达到127.5 Iu/M l。Endo-1,4-xylanase( E.C.3.2.1.8) is the major enzyme to the conversion of hemicelluloses into xylo-oligosaccharide.In this research,a novel GH10 xylanase Ⅲ( xyn Ⅲ) gene was cloned from Hypocrea orientalis EU7-22 by chromosome walking and PCR.The results showed that the DNA fragment( 1283 bp) encoding xynⅢ( gxynⅢ) contained three introns.The CDS of xynⅢ( cxynⅢ) encoded 331 amino acids of putative mature protein and a 16 aa signal in N terminator.The amino acid sequence of xynⅢ is highly homologous with the endoxylanase of Trichoderma pseudokoningii.The bioinformatics analysis showed that the theoretical isoelectric point and the molecular weight of putative mature protein of XYNⅢ were 6.14 and 36.55 ku,respectively.It is a soluble hydrophilic protein containing 18 N-glycosylation sites.The 3D structure predicted with SWISS-Model showed that XYNⅢ protein contained11 alpha helices and 8 extended strands.A recombinant plasmid p PIC9K-xynⅢ was constructed and then transformed into Pichia pastoris.The transformant identified by PCR was induced to produce XYNⅢ enzyme with 1% methanol.And after 168 hours induced expression,the produced crude enzyme was detected to reach a high enzymatic activity of 127.5 IU / m L.国家自然科学基金(21303142;31170067); 福建省海洋高新产业发展专项项目(闽海洋高新[2014]25号); 厦门市海洋经济发展专项资金项目(14GZP59HJ29

Variation of Yield and Composition of Energy Plant Erianthus arundinaceum During Different Growth Stages

研究不同时期斑茅产量及其化学组分的变化。结果表明:斑茅生物质产量在11月达到最高,其后开始下降;整个生长发育过程中茎叶生物质产量比呈现上升趋势;含水量随生长发育的进行显著降低;叶片灰分含量全年最低为5月,从10月份开始显著增加,至12月份达到最高。茎秆灰分含量略低于叶片,全年最低为10月,最高为7月,变化幅度不大。叶片中木质素含量全年由高到低排列顺序为12月>11月>9月>10月>7月>8月>6月;茎秆中木质素含量全年由高到低排列顺序为12月>11月>9月>7月>8月>10月>6月。10月份茎秆含水量低,灰分含量最低,热值较高,结合产量和燃料品质考虑其更适于直接燃烧,而9月份叶片则更加适于作为生物转化的材料。The effect of harvesting time on the yield and chemical composition of E.arundinaceum was studied.The results revealed that the highest biomass yield was available in November, and then the proportion of leaf biomass was gradually started to decline.The water content decreased significantly while the quantity of ash in leaves remained the lowest in May, which increased significantly in October and reached the highest amount in December.The quantity of ash in the stem was slightly lower than that in the leaf with little change year around.The maximum was appeared in July and the minimum was observed in October.The lignin content in the leaf was in the following downward order: Dec., Nov., Sept., Oct., July, Aug., June, and that for the stem was: Dec.,Nov., Sept., July., Aug., Oct., June.The calorific value of the leaf was the lowest in September and the highest in October, whereas that for the stem was the highest in August.The study showed that the best biomass accumulation was usually available with the lowest moisture and ash in October, which was more suitable for the production of fuel.The results proved that the lignin content of the leaf was lower than that of stem, which may be more suitable for bioconversion.国家自然科学基金(No.31170067;No.21303142); 福建省自然科学基金(No.2012J05029); 国家重点基础研究发展计划(973计划)(No.2010CB732201

HPLC and MALDI-Tof-MS Analysis of Hydrolysates of Xylan

利用HPlC结合MAldI-TOf-MS对1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生化后山毛榉木聚糖水解产物进行分析,检测到了难以获得标准品对照的木聚糖水解产物。结果表明,稀硫酸水解山毛榉木聚糖的主要水解产物有木糖和4-O-甲基葡萄糖醛酸-木糖(b2),以及少量4-O-甲基葡萄糖醛酸(b1)。内切重组木聚糖酶An Xyn10C水解山毛榉木聚糖产生木糖、木二糖和4-O-甲基葡萄糖醛酸-木三糖(b3),而内切重组木聚糖酶HO Xyn11A水解山毛榉木聚糖主要产生木糖、木二糖、木三糖、4-O-甲基葡萄糖醛酸-木四糖(b4)和4-O-甲基葡萄糖醛酸-木五糖(b5)。基于PMP柱前衍生化的HPlC结合MAldI-TOf-MS方法能高效地分析复杂的木聚糖水解产物。The hydrolysis end products of beechwood xylan,which were released by sulfuric acid or enzymes and then labeled at their reducing ends with 1-phenyl-3-methyl-5-pyrazolone( PMP) derivatization,were analyzed by HPLC assisted with MALDITof-MS.Some of the hydrolysates,which were lack of related commercial available standard substances,were determined.It was found that the xylose and 4-O-methyl-glucuronic acid-xylose( B2) were the main products with minor amounts of 4-Omethylglucuronic acid( B1) in the hydrolysates of beechwood xylan by sulfuric acid.Recombinant endo-β-1,4-xylanase An Xyn10 C released xylose,xylobiose,and 4-O-methyl-glucuronic acid-xylotriose( B3) as the main hydrolysates from beechwood xylan,whereas recombinant endo-β-1,4-xylanase Ho Xyn11 A released xylose,xylobiose,xylotriose,4-O-methyl-glucuronic acidxylotetrose( B4) and-xylopentaose( B5),and aldohexaouronic acid.These results revealed that HPLC assisted with MALDITof-MS based on PMP derivatization was a very useful and robust method for the determination of products in hydrolysis of xylan.国家自然科学基金资助项目(31170067); 厦门市海洋经济发展专项资金项目(14GZP59HJ29); 福建省海洋高新产业发展专项项目(闽海洋高新[2014]25号